This renewal application is focused on understand the molecular mechanisms of hepatic innate immunity that controls the outcome of hepatitis C virus (HCV) infection. We have shown that retinoic acid inducible gene-I (RIG-I) initiates the hepatic innate immune response upon recognition and binding of the pathogen associated molecular pattern (PAMP) consisting of 5' triphosphate with poly-uridine (poly-U/UC) within the HCV RNA. During acute infection this recognition of transmission/founder (T/F) viral genome by RIG-I results in its modification and activation to bind the MAVS adaptor protein on mitochondria-associated membranes (MAM) and signal hepatic innate immune defenses that restrict infection. Our preliminary studies now reveal that T/F genomes from patients exhibit remarkable variation within their poly-U/UC motif, and that this variation imparts differential recognition and binding by RIG-I that allows escape from non-self recognition. Moreover, we have found that signaling by RIG-I is dependent on formation of a RIG-I translocon protein complex to facilitates RIG-I cytosol-to-membrane translocation for MAVS interaction and signaling. Our studies now reveal that these activities are governed by the reversible acteylation of RIG-I, and that this process is likely controlled through the HDAC6 protein as a RIG-I cofactor of innate immunity. We show that HCV evades hepatic innate immunity via MAM targeting by the NS3/4A viral protease to cleave MAVS and disrupt RIG-I signaling, thereby facilitating chronic infection, and this action is dependent on stable NS3/4A-MAVS interaction on the MAM. Ablation of this interaction restores innate immunity to prevent acute to chronic HCV transition. We hypothesize that regulation of the RIG-I pathway and hepatic innate immunity are major determinants controlling the acute to chronic transition of HCV infection. We will therefore conduct studies to: 1) Define the features of RIG-I and the HCV poly-U/UC motif that confer PAMP binding and innate immune signaling activation, and determine how PAMP variation among T/F genomes influences acute innate immune induction by HCV; 2) Determine the role of reversible acetylation in RIG-I translocon assembly and function; 3. Identify the cognate/reciprocal motifs of NS3/4A and MAVS that mediate interaction and control of the RIG-I pathway.